The use of immobilized enzyme catalysts either "as is" or in their intracellular form has become an integral aspect of industrial chemical production. Immobilized catalysts have been developed for the production of many chemical and food products, and include enzymic systems such as aspartase, penicillin acylase, glucose isomerase, beta-galactosidase, alpha-amylase, and amino acylase. The immobilization of such catalysts has generally included methods such as entrapment within cross-linked gels; encapsulation within hollow fibers/macro-capsules; adsorption on inert supports/ion-exchange resins; cross-linking by multifunctional reagents; and covalent binding to polymeric supports. Examples of these various methods and their application abound in the literature and review articles (e.g. Applied Biochemistry and Bioengineering, Vol. 1, Immobilized Enzyme Principles) and will not be discussed here. Industrial applications of these various methods have had to focus on the feasibility of the selected support matrix for large-scale operations. It is particularly important for industrial applications that the matrix have good mechanical stability and good hydrodynamic properties such that compression, compaction, and/or breaking of the support does not occur upon extended use. The support must also be of suitable permeability and surface area and of such a structure that immobilized catalyst activity is maximized and diffusional resistances are minimized.
In answer to these requirements, Chibata et al (U.S. Pat. No. 3,791,926) have developed various methods of entrapping enzymes/microorganisms within synthetic-type polymer matrices (e.g. polyacrylamide), particularly for the production of aspartic acid. Nelson (U.S. Pat. No. 3,957,580) has similarly reported a method of entrapping/cross-linking enzyme-containing microbial cells within other types of synthetic polymer systems in which the immobilized cells are further cross-linked to the polymer matrix by poly-functional reagents such as glutaraldehyde. Synthetic polymer systems of these types have two major drawbacks: (1) the preparation of the immobilized catalyst can involve the use of toxic irritants (monomers, initiators, cross-linkers, etc.) which would present problems in the production of food-grade products; and (2) the support matrix can deform/or compact upon extended use in a large-scale column reactor system.
To improve the characteristics of gel-entrapped immobilized catalysts, Chibata et al have investigated the use of sulfated polysaccharide gels (such as kappa-carrageenan) as a support matrix (U.S. Pat. No. 4,138,292). These types of gels can be used in a variety of configurations (beads, membranes, etc.) for the immobilization of enzymes and microorganisms. The limitation in these applications is that the polysaccharide must contain 10% (w/w) sulfate moiety, and that a gel-hardening reagent (e.g. a water-soluble organic amine or a metal ion of atomic weight greater than 24) must be used to ensure a stable gel support. Previous literature reports have indicated that microorganisms entrapped with an agar gel matrix (Japan Patent Application No. 95470/1975) did not retain the gel shape, particularly at temperatures above 40.degree. C., and was transformed into a "sol" structure. Similarly, carageenan exhibits this same loss of form/structure unless gel-hardening reagents of the type mentioned above are employed to retain mechanical stability. I have now found that stable, catalytically active systems can be obtained using an agar gel containing less than 10% (w/w) sulfate moiety which does not require any of the previously used gel-hardening reagents. Furthermore, this type of system can be used in the form of a "fiber catalyst" in a column reactor and not lose its shape. The illustrative example with this newly developed immobilization process utilizes an aspartase-containing microorganism which is used as an immobilized cell column reactor for the continuous-flow production of L-aspartic acid from an ammonium fumarate substrate. The interest in this particular immobilized cell system has to do with the importance of L-aspartic acid itself, which is used as a food-grade product and as an intermediate in the production of other food-grade products. Industrial production of aspartic acid has generally involved either batch-fermentation methods in which aspartase-containing microorganisms are used to carry out the reaction and are then discarded after a single use, or have involved the use of immobilized aspartase-containing microorganisms which may be used repeatedly in a continuous-flow mode of operation. Due to the reduced labor costs and reuseable form of the catalyst associated with the latter method, it is generally favored over the batch-type systems provided that a suitably designed (good activity, stability, etc.) immobilized cell catalyst is available. The present invention does allow the production of such an immobilized cell derived catalyst.
The methodology employed in this invention is adaptable for other immobilized cell processes (e.g. penicillin acylase systems), and also to native enzyme immobilization: the exact application(s) will depend on the requirements of the practitioner of the invention.